Over the course of the last decade, bioorthogonal chemical methods have been developed for the site-specific chemical modification of proteins and used to alter their properties and function.1-7 For example, fluorophores can be site-specifically attached to proteins as a biophysical or cellular localization tool, while protein-polymer conjugation is a well-established method for modulating the in vivo behavior of proteins.8-11 In addition, a number of groups have reported bioorthogonal approaches for the construction of bifunctional protein assemblies. Schultz and co-workers coupled two antibody FABs via an alkyne-azide cycloaddition click reaction using non-natural mutagenesis techniques.12 Bertozzi and coworkers, used an enzymatic formyl generating strategy13 to generate an aldehyde that was then converted to a cyclooctyne- or azide-functionalized protein via oxime formation followed by reaction with other azide-modified peptides or proteins. Ploegh and coworkers used a variation of sortagging to create N-to-N and C-to-C protein conjugates by preparing pairs of azide- and alkyne-containing proteins that were then linked via click reactions.14 In the above examples, proteins equipped with a single bioorthogonal group were modified with a second small molecule, polymer or protein bearing a complementary functional group.
Recently, progress towards the introduction of multiple functional groups into proteins has also been made. Wu and coworkers developed a strategy for site-specific two-color labeling of a Rab GTPase for FRET applications by applying chemoselective native chemical ligation and oxime ligation simultaneously.15 A C-terminal oxime was generated via expression of a C-terminal thioester while an N-terminal cysteine (for subsequent ligation) was revealed by TEV-catalyzed proteolysis. In other work, Schultz and coworkers developed a method for site-specific dual-labeling of proteins for FRET analysis based on the use of selective cysteine alkylation combined with non-natural amino acid incorporation of a ketone moiety.16 Park and coworkers successfully incorporated two unnatural amino acids bearing ketone and alkyne groups into a protein for analysis of protein dynamics using a related nonsense suppression approach.17 Very recently, Chen and coworkers, designed and synthesized bifunctional sialic acid analogues containing azide and alkyne moieties for incorporation of two distinct chemical reporters into cellular sialylated glycans for FRET imaging.18 While useful, that method is limited to sialylated-cell surface glycans and requires metabolic activation of the bifunctional sialic acid analogue to the corresponding CMP-sugar prior to incorporation.
Accordingly it would be desirable to provide new reagents for introducing multiple functional groups into proteins.